The permeability of mitochondrial outer membrane (MOM) is regulated by the proteins of the Bcl-2 family via their interactions at the membrane. While pro-apoptotic Bax protein promotes MOM permeabilization (MOMP) releasing cytochrome c after activation by BH3-only protein, anti-apoptotic Bcl-2 protein protects MOM. However both Bax and Bcl-2 can form pores in model membranes. Unlike Bax pore that has been extensively studied and reported to be directly linked to MOMP, Bcl-2 pore is much less known; thus we investigated the pore-forming property of recombinant Bcl-2 lacking the C-terminal transmembrane sequence (Bcl-2deltaTM) in liposomal membranes of MOM lipids. We found that: (1) Bcl-2 formed pores at acidic pH that induced the association of Bcl-2 with liposome; (2) Bcl-2 pore size was dependent on Bcl-2 concentration, suggesting that oligomerization is involved in Bcl-2 pore formation; (3) Unlike Bax pore that could release large molecules up to 2 mega-Da, Bcl-2 pore was smaller and could only release the molecules of a few kilo-Da. Therefore, Bcl-2 and Bax may form different size pores in MOM, and while the large pore formed by Bax may release cytochrome c during apoptosis, the small pore formed by Bcl-2 may maintain the normal MOM permeability.
BH3 Interacting Domain Death Agonist Protein ; metabolism ; Cell Membrane Permeability ; Cytochrome c Group ; metabolism ; Humans ; Hydrogen-Ion Concentration ; Liposomes ; metabolism ; Mitochondrial Membrane Transport Proteins ; metabolism ; Mitochondrial Membranes ; metabolism ; bcl-2-Associated X Protein ; metabolism ; bcl-X Protein ; metabolism

The purpose of study was to explore the possible functions of Bcl-xL in the glucosamine sulfate-induced apoptosis of chronic myeloid leukemia K562 cells. Light microscopy and Wright-Giemsa staining were used to investigate the morphologic evidences for apoptosis of K562 cells induced by glucosamine sulfate (GS); immunofluorescence was used to observe the translocation of cathepsin D and cytochrome C during the apoptosis; Western blot was performed to detect the expression of Bcl-xL, Bid, Bax in K562 cells treated by GS. The results showed that many vacuoles were observed in the cytoplasma of the K562 cells treated by GS; fluorescent signals of cathepsin D and cytochrome were fransformed from granules to disperse form by using immunofluorescence; the expression of Bcl-xL was found down-regulated in K562 cells treated by GS, but not in the cells pre-treated with pepstatin A; the significant changes were not detected in expression of Bax and Bid protein before or after apoptosis. It is concluded that Bcl-xL protein may mediate relationship between cathepsin D and mitochondia pathway, Cathepsin D may play an important role in the GS inducing apoptosis of K562 cells through downregulation of Bcl-xL expression.
Apoptosis ; drug effects ; physiology ; BH3 Interacting Domain Death Agonist Protein ; metabolism ; Blotting, Western ; Cathepsin D ; metabolism ; Cytochromes c ; metabolism ; Fluorescent Antibody Technique ; Glucosamine ; pharmacology ; Humans ; K562 Cells ; bcl-2-Associated X Protein ; metabolism ; bcl-X Protein ; metabolism ; physiology

AIMTo investigate apoptosis induced by 3,3'-diethyl-9-methylthia-carbocyanine iodide (DMTCCI), an inhibitor of DNA primase found in our previous study, and the mechanism of DMTCCI in human myelogenous leukemia HL-60 cells.

METHODSHL-60 cells were cultured in RPMI-1640 medium and treated with different concentrations of DMTCCI. MTT assay was used to detect growth inhibition. Flow cytometry and DNA ladders were used to detect apoptosis. Western blotting was used to observe the expression of survivin, Bcl-xL, Bad, Bax, Bcl-2, caspase-9, caspase-3, caspase-6, PARP, DFF45 and lamin B protein. Caspase-3 activity was measured by ApoAlert Caspase-3 Assay Kit.

RESULTSDMTCCI inhibited proliferation of human leukemia HL-60 cells with IC50 value of 0.24 micromol x L(-1). The results of flow cytometry and DNA ladders showed that DMTCCI could induce apoptosis of HL-60 cells. The expression levels of protein survivin and Bcl-xL were down-regulated, Bad and Bax were up-regulated, while Bcl-2 protein had no change in response to DMTCCI treatment in HL-60 cells. Treatment of HL-60 cells with DMTCCI induced the proteolytic cleavage of caspase-9, caspase-3, caspase-6, PARP, DFF45 and lamin B protein. Caspase-3 activity apparently increased at 3 h and reached a peak at 12 h after exposure to 1 micromol x L(-1) of DMTCCI in HL-60 cells.

CONCLUSIONDMTCCI inhibited proliferation and induced apoptosis of human leukemia HL-60 cells. Bcl-2 family proteins, survivin and caspases family proteins might play a role in the apoptosis process induced by DMTCCI.

Apoptosis ; drug effects ; Carbocyanines ; pharmacology ; Caspase 3 ; metabolism ; Cell Proliferation ; drug effects ; DNA Damage ; DNA Fragmentation ; drug effects ; DNA Primase ; antagonists & inhibitors ; Flow Cytometry ; HL-60 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; Leukemia, Myeloid ; metabolism ; pathology ; Microtubule-Associated Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; bcl-2-Associated X Protein ; metabolism ; bcl-Associated Death Protein ; metabolism ; bcl-X Protein ; metabolism

OBJECTIVETo study the effects of sodium butyrate (NaBu) on cell cycle checkpoint and the apoptosis sensitivity in U937 cells.

METHODSTwo mutant U937 cell lines, U937-ASPI3K (ATM negative) and U937-pZeosv2(+) (ATM wild-type), were used as the cell model system. Immunoprecipitation and kinase assay were used to examine the p38 MAPK and ERK1 kinase activities. Western blot was used to analyze the phosphorylation of Bad protein.

RESULTSU937-pZeosv2(+) pretreated with NaBu exhibited enhanced apoptotic response in a NaBu dose dependent fashion upon (137)Cs irradiation, which could be abolished by olomoucine (OLM), a p38 MAPK specific inhibitor. On the other hand, Cyclin dependent kinase 2 (CDK2) specific inhibitor CDK2-I and p34cdc2/cyclinB inhibitor alsterpaullone (ALP) failed to block the effects of NaBu. Similar results were also observed in U937-ASPI3K. The effect of irradiation on p38 MAPK and ERK1 was strikingly potentiated by NaBu. Furthermore, inactivation of irradiated Bad protein via phosphorylation on serine 136 was also enhanced.

CONCLUSIONNaBu is able to enhance the apoptotic response in U937 cells, which is mediated by p38 MAPK activation but not ATM status.

Apoptosis ; Butyrates ; pharmacology ; Carrier Proteins ; metabolism ; Humans ; MAP Kinase Signaling System ; Mitogen-Activated Protein Kinase 3 ; Mitogen-Activated Protein Kinases ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; U937 Cells ; bcl-Associated Death Protein ; p38 Mitogen-Activated Protein Kinases

OBJECTIVETo deeply explore the effects of microcystins (MC-LR) on Bax and Bcl-2 during the course of MC-LR promoting liver tumor.

METHODSapplied to set up the animal model, and the effect of MC-LR promoting liver tumor was evaluated by the Albertgamma-GT methods. And then, the immunohistochemical technique, RT-PCR and image analysis were used to study the expression of the Bcl-2 and Bax during the course of promoting tumor.

RESULTS(1) MC-LR might enhance the positive reaction rate of GGT. The positive reaction rate of GGT in DEN + pure toxin group was 100%, it was significantly higher than the DEN control group 22.22% (P < 0.05). (2) The intension and areas of the protein expression of Bcl-2 in DEN + pure toxin group were 0.0977 and 0.0315, and in DEN control group were 0.0460 and 0.0205, respectively. The expression level of Bcl-2 protein in DEN + pure toxin group were significantly higher than in DEN control group (P < 0.05). Simultaneously, the protein expression of Bax was significantly decreased by MC-LR (P < 0.05). The intension and areas of the expression of Bax in DEN + pure toxin group were 0.0283 and 0.0073, and in DEN control group were 0.0655 and 0.0244 respectively. (3) The mRNA expression of Bcl-2 was significantly increased by MC-LR. The intension of Bcl-2 mRNA expression in DEN + pure toxin group was 2.244, being significantly higher than in the other groups (P < 0.05). However, the mRNA expression of Bax showed no significant difference between DEN + pure toxin and the other groups.

CONCLUSIONThe expression change of Bcl-2 and Bax should possibly play an important role in the course of MC-LR promoting liver tumor.

Animals ; Apoptosis ; Carcinogens ; toxicity ; Hepatocytes ; metabolism ; Liver Neoplasms ; chemically induced ; metabolism ; Male ; Microcystins ; toxicity ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; bcl-2-Associated X Protein ; biosynthesis ; bcl-Associated Death Protein ; biosynthesis

Adult ; Altitude Sickness ; metabolism ; pathology ; Apoptosis ; BH3 Interacting Domain Death Agonist Protein ; metabolism ; Erythroblasts ; cytology ; pathology ; Humans ; Male ; Middle Aged ; RNA, Messenger ; genetics ; bcl-2-Associated X Protein ; metabolism ; bcl-X Protein ; metabolism

OBJECTIVETo evaluate the effects of valsartan and carnitine on cardiomyocyte Calpain-1 and Bcl-xl expressions of dogs with chronic alcohol intake-induced cardiomyopathy.

METHODSDogs were randomly assigned into 4 groups (n = 7 each): (1) alcohol fed (free access to 5%, 1(st) week; 10% 2(nd) week; 500 ml 25% bolus plus free access to 5% from 3 to 24 weeks, A); (2) alcohol + valsartan (5 mg×kg(-1)×d(-1), B); (3) alcohol + carnitine (300 mg×kg(-1)×d(-1), C); (4) Control (D). After six months, all animals were assessed for left ventricular (LV) function by echocardiography. The Bad and Bcl-xl protein expressions were evaluated by immunohistochemistry. The expression of Calpain-1 protein was determined with Western blot. Myocardial morphology was quantified on HE stained slices and under electron microscopy. The terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) was performed for apoptosis analysis.

RESULTSCompared with group D, LVEDD and LVESD were significantly increased while EF and FS significantly decreased in group A. In alcohol fed group, expressions of Bad and Calpain-1 protein were significantly increased while Bcl-xl protein expression was downregulated, all changes could be significantly attenuated by intervention with valsartan and carnitine (all P < 0.05).

CONCLUSIONThese data suggest that alcohol could promote cardiac myocyte apoptosis, reduce cardiac function and aggravate myocardial remodeling which valsartan and carnitine could reduce alcoholic cardiomyopathy by downregulating Calpain-1 and Bad protein expression and upregulating expression of Bcl-xl protein.

Animals ; Apoptosis ; drug effects ; Calpain ; metabolism ; Cardiomyopathy, Alcoholic ; metabolism ; pathology ; Carnitine ; pharmacology ; Disease Models, Animal ; Dogs ; Myocytes, Cardiac ; drug effects ; metabolism ; Tetrazoles ; pharmacology ; Valine ; analogs & derivatives ; pharmacology ; Valsartan ; bcl-Associated Death Protein ; metabolism ; bcl-X Protein ; metabolism

OBJECTIVETo explore the molecular mechanisms by which mitochondrial estrogen receptor β (ERβ) suppresses non-small cell lung cancer cell apoptosis induced by apoptotic stimulations.

METHODSThe mitochondrial localization of ERβ in non-small cell lung cancer cell lines A549 and 201T was determined using immunofluorescence and Western blotting. The changes of apoptosis of the cells with mitochondrial ERβ overexpression or knockdown in response to cisplatin and STS treatments were assessed, and mitochondrial ERβ interaction with the pro-apoptotic protein Bad was detected using co-immunoprecipitation and Western blotting.

RESULTSERβ was localized in the mitochondria in A549 and 201T cells. ERβ overexpression significantly reduced while ERβ knockdown increased Bax activation and cell apoptosis induced by cisplatin and STS. Mitochondrial ERβ interaction with pro-apoptotic protein Bad may suppress Bax activation and its translocation to the mitochondria.

CONCLUSIONMitochondrial ERβ can suppress apoptosis of non-small cell lung cancer cells induced by cisplatin or STS through interaction with Bad, suggesting the value of mitochondrial ERβ as a new therapeutic target for treatment of non-small cell lung cancer.

Apoptosis ; Carcinoma, Non-Small-Cell Lung ; pathology ; Cell Line, Tumor ; Cisplatin ; Estrogen Receptor beta ; metabolism ; Humans ; Mitochondrial Proteins ; metabolism ; bcl-Associated Death Protein ; metabolism

OBJECTIVETo investigate the role of reactive oxygen species (ROS) and ROS-caused mitochondrial transmembrane potential loss in sodium selenite-induced apoptosis in NB4 cells.

METHODSROS production was measured by ROS-specific probe DCFH-DA. Sodium selenite mitochondrial transmembrane potential loss was evaluated by flow cytometry with Rh123 staining. Protein levels of cytochrome C, Bid, Bcl-xl, and Bax were measured by Western blot using protein-specific antibodies. NB4 cells were pre-incubated by MnTmPy or BSO before selenite treatment to further confirm the effects of ROS on NB4 cells.

RESULTS20 micromol/L sodium selenite induced ROS production and mitochondrial transmembrane potential loss in NB4 cells time-dependently. Cytochrome C accumulated in cytoplasm after selenite treatment. Sodium selenite also downregulated Bcl-xl and activated Bax and Bid at protein level. Pretreatment with antioxidant MnTmPy almost fully abrogated the proapoptotic effect of sodium selenite prevented the cleavage of Bid protein and in turn the mitochondrail transmembrane potential loss. On the contrary, pretreatment with BSO intensified the mitochondrail transmembrane potential loss induced by sodium selenite.

CONCLUSIONSSodium selenite may induce apoptosis by inducing ROS production in NB4 cells, which leads to the downregulation of Bcl-xl, upregulation of Bax, and cleavage and activation of Bid. Bax and tBid then agregate on mitochondrial membrane, which in turn causes a decrease of mitochondrial transmembrane potential and release of cytochrome C into cytoplasm.

Apoptosis ; BH3 Interacting Domain Death Agonist Protein ; biosynthesis ; Cell Line, Tumor ; Cytochromes c ; metabolism ; Humans ; Membrane Potential, Mitochondrial ; drug effects ; Reactive Oxygen Species ; metabolism ; Sodium Selenite ; pharmacology ; bcl-2-Associated X Protein ; biosynthesis ; bcl-X Protein ; biosynthesis

PURPOSE: To investigate the anti-tumor effect of capsaicin on human pharyngeal squamous carcinoma cells (FaDu). MATERIALS AND METHODS: The expression of apoptosis/cell cycle-related proteins (or genes) was examined by reverse transcriptase-polymerase chain reaction, western blotting and ELISA methods, while the apoptotic cell population, cell morphology and DNA fragmentation levels were assessed using flow cytometry, fluorescence microscopy and agarose gel electrophoresis. RESULTS: Capsaicin was found to inhibit the growth and proliferation of FaDu cells in a dose- and time-dependent manner. Apoptotic cell death was confirmed by observing increases in nuclear condensation, nuclear DNA fragmentation and sub-G1 DNA content. The observed increase in cytosolic cytochrome c, activation of caspase 3 and PARP (p85) levels following capsaicin treatment indicated that the apoptotic response was mitochondrial pathway-dependent. Gene/protein expression analysis of Bcl-2, Bad and Bax further revealed decreased anti-apoptotic Bcl-2 protein and increased pro-apoptotic Bad/Bax expression. Furthermore, capsaicin suppressed the cell cycle progression at the G1/S phase in FaDu cells by decreasing the expression of the regulators of cyclin B1 and D1, as well as cyclin-dependent protein kinases cdk-1, cdk-2 and cdk-4. CONCLUSION: Our current data show that capsaicin induces apoptosis in FaDu cells and this response is associated with mitochondrial pathways, possibly by mediating cell cycle arrest at G1/S.
Apoptosis/drug effects ; Blotting, Western ; Capsaicin/*pharmacology ; Carcinoma, Squamous Cell/*metabolism ; Cell Cycle/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Humans ; Microscopy, Fluorescence ; Pharyngeal Neoplasms/*metabolism ; Proto-Oncogene Proteins c-bcl-2/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; bcl-2-Associated X Protein/genetics/metabolism ; bcl-Associated Death Protein/genetics/metabolism